Medical Mycology December 2008, 46, 811822
Diagnostic value of morphological, physiological and
biochemical tests in distinguishing Trichophyton rubrum
from Trichophyton mentagrophytes complex
AYLİN ATES, KADRİ OZCAN & MACİT ILKIT
Department of Microbiology, Faculty of Medicine, University of Cukurova, Adana, Turkey
Keywords Dermatophytes, Lowenstein-Jensen
grophytes, Trichophyton rubrum, urea Petri agar
Introduction
Currently there has been a rapid and striking change in
dermatophyte taxonomy as a result of the introduction
of new techniques such as the sequencing of the
Received 26 February 2008; Accepted 7 April 2008
Correspondence: Macit Ilkit, Division of Mycology, Department of
Microbiology, Faculty of Medicine, University of Cukurova, Adana,
01330, Turkey. Tel: 90 532 286 00 99; Fax: 90 322 457 30 72;
E-mail: [email protected]
– 2008 ISHAM
agar,
Trichophyton
menta-
internal transcribed spacer region. Most of the earlier
varietal names were found to have been invalidly
described or to have been reported on the basis of
minor characteristics such a color variations in their in
vitro appearance. Furthermore, some of these names
are now considered to represent species rather than
varieties [15]. In other studies volatile fingerprints [6]
and enzyme activities [7] were used with significant
success in the rapid identification of Trichophyton spp.
In most mycology laboratories, these keratinophilic
fungi are identified to species on the basis of colony
DOI: 10.1080/13693780802108458
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
The two most frequently encountered dermatophyte etiologic agents of glabrous
skin and nail dermatophytoses are Trichophyton rubrum and T. mentagrophytes.
This study was aimed to discuss the efficacy of morphological, physiological and
biochemical diagnostic tests commonly used in the identification of T. rubrum and
members of the T. mentagrophytes complex. In this study, we evaluated; hydrolysis
of urea in broth and on urea agar slants and Petri plates incubated at 228C, 288C
and 378C, in vitro hair perforation (blond child, sheep and goat hair), pigment
production on cornmeal dextrose agar (CMDA) and bromcresol purple-milk
solids-glucose agar (BCP-MS-G), Tween opacity, sorbitol assimilation, and salt
tolerance. Additionally, the production of micro- and macroconidia was investigated by using brain heart infusion agar (BHIA), Christensen’s urea agar in Petri
plates (UPA), CMDA, Lowenstein-Jensen agar (LJA), malt extract agar, oatmeal
agar, Oxoid chromogenic Candida agar, and potato dextrose agar. All cultures were
incubated at 288C, and conidial production was compared on days 5, 10 and 15. It
was found that the urea hydrolysis test yielded more rapid and significant results
when urea medium was prepared in Petri plates and incubated at 288C (PB0.01).
LJA supported the highest production of microconidia after 15 days (P B0.001).
Additionally, it was found that T. rubrum strains produced red pigment on CMDA
(PB0.01) and BCP-MS-G, while strains of the T. mentagrophytes species complex
did not. A special algorithm containing the various test procedures employed in
these studies is presented which was found to be useful in the differentiation of T.
rubrum strains from T. mentagrophytes complex. Our results revealed that UPA,
CMDA, BCP-MS-G, LJA, and BHIA may be used as common mycological agars
in routine practice.
812
Ates et al.
Material and methods
Fungal strains
Table 1 lists the 18 T. rubrum isolates, 12 members of
the T. mentagrophytes complex and 4 Arthroderma
strains used in this study, as well as their origins and
reference numbers in the culture collection of the
Centraalbureau voor Schimmelcultures (CBS),
Utrecht, the Netherlands. All strains were subcultured
on Sabouraud’s glucose agar (SGA, Merck, Darmstadt, Germany) at 268C for 3 weeks. All tests were
carried out from August 2006 to December 2007 at the
Division of Mycology, Department of Microbiology,
Faculty of Medicine, University of Cukurova.
Hydrolysis of urea
The ability to hydrolyse urea provides additional data
to aid in distinguishing the typical, cosmopolitan form
of T. rubrum (urease-negative) from; (1) members of the
T. mentagrophytes complex (typically urease-positive,
except T. erinacei known as hedgehog fungus), (2) the
urease-positive ‘Afro-Asiatic’ or ‘granular’ variety of T.
rubrum, formerly often called T. raubitschekii, and (3)
T. rubrum with ‘megninii’ morphotype [811,14,15]. In
general, T. mentagrophytes hydrolyses urea ( 57 days)
much earlier than T. rubrum (7 days) [15]. In this
study, Christensen’s urea agar (Oxoid, Hampshire,
England) both in slants and in Petri plates, as well as
urea broth were used. After the urea media were
inoculated, the cultures were incubated at 228C, 288C,
and 378C for up to 4 weeks. The color change of the
media from orange or pale pink to purple-red indicated
positive results, i.e., the presence of urease. The data
from the three different media and three incubation
temperatures were compared with each other. In
addition, the micromorphology of colonies on urea
Petri agar (UPA) were examined.
In vitro hair perforation test
This test distinguishes between atypical isolates of the
T. rubrum and T. mentagrophytes complex. Hairs
exposed to T. mentagrophytes complex members show
wedge-shaped perforations perpendicular to the hair
shaft (a positive test result), whereas isolates of T.
rubrum lack this ability to form perforations. Short
strands of blond hair from children under 5 years of
age, as well as hair samples from sheep and goats were
placed in Petri plates and autoclaved at 1218C for
10 min. Twenty-five ml of sterile distilled water and 2 or
3 drops of a sterile solution of 10% yeast extract were
then added to the plates. The plates were inoculated
– 2008 ISHAM, Medical Mycology, 46, 811822
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characteristics, microscopic morphologies, growth requirements, and physiological and biochemical test
results [811]. Due to the resources requirements of
various molecular identification procedures and equipment, they are of little practical use except in mid-tohigh level reference laboratories [11]. Furthermore, as
changes are observed for a single dermatophyte species
in its morphology (colony pattern, pigments and
growth rate) and physiology (hydrolysis of urea and
in vitro hair perforation), as well as in its biochemistry
(sorbitol assimilation) and genotype (rRNA and mating patterns), the reliability of the conventional methods noted above are questioned [811]. Moreover, such
isolates are problematic with respect to their proper
identification in a clinical laboratory. Many dermatophyte species maintained under laboratory conditions
lose their original colonial features and ability to form
macro- and microconidia. This is especially true for
Trichophyton rubrum and members of the T. mentagrophytes complex when recovered from chronic infections
being treated with various antifungal agents as they
often do not manifest their typical colonial morphology, pigmentation and production of micro- and
macroconidia.
Recent studies have demonstrated that T. rubrum is
composed of two anamorphic taxa, i.e., T. rubrum, and
T. violaceum and that genotypes of T. rubrum show a
worldwide distribution excluding the African continent
[4,5]. On the other hand, such microorganisms like
T. rubrum with fischeri, kanei, and raubitschekii morphotypes are genetically indistinguishable [4]. Gräser
et al. [12] studied a set of T. rubrum strains including
phenotypic varieties like T. rubrum var. nigricans, and
by using molecular tools, could not detect any DNA
differences among the varieties. On the other hand, the
T. mentagrophytes complex is a group of related species
formerly identified under this familiar name, but is now
distinguishable through sequence-based studies into
three distinct phylogenetic complexes, i.e., Arthroderma
vanbreuseghemii/T. interdigitale, A. benhamiae/T. erinacei and T. mentagrophytes per se (now taxonomically
classified as the former T. mentagrophytes var. quinckeanum) [2,3,13].
One of the prominent problems observed in mycology laboratories is distinguishing T. rubrum strains
from members of the T. mentagrophytes species complex by using conventional diagnostic methods. The
present study aimed to (i) discuss morphological,
physiological and biochemical procedures used in the
identification of both species, and (ii) measure the
reliability of such procedures in routine laboratory
practices.
Conventional diagnosis of Trichophyton spp.
813
Table 1 Strains analysed in this study.
Reference no.
Strain-dependent deviation Clinical picture
Geography
T . rubrum
T. fischeri
T. fluviomunionse
T. kanei
T. kuryangei
T. kuryangei
T. kuryangei
T. megninii
T. pervesii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. rodhainii
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. r. var. nigricans
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
100081
592.68
289.86
517.63
518.63
422.67
389.58
303.38
202.88
287.86
100084
102856
376.49
363.53
392.58
302.60
363.62
100237
Abundant sporulation
Microconidia absent
LHistidine requirement
Abundant MA; urease Abundant MA; urease Abundant MA; urease Abundant MA; urease Melanoid pigmentation
Contaminant
Human, skin
Human, buttock
Child, tinea capitis
Human, tinea capitis
Man, nail
Child, tinea capitis
Human, tinea pedis
Human, skin
Human, skin
Human thumb nail
Human, tinea cruris
Man
Human, tinea pedis
Man, skin
Man, nail
Rio Muni, Guinea
Ontario, Canada
Congo
Zaire
Utrecht, Netherlands
D’Adrar Sahara, Mauretania
Ontario, Toronto, Canada
Toronto, Canada
Canada
Cameroon, Italy
Congo
Rotterdam, Netherlands
Netherlands
Netherlands
T. mentagrophytes complex
T. asteroides
T. erinacei
T. erinacei
T. erinacei
T. interdigitale
T. interdigitale
T. langeronii
T. mentagrophytes
T. m.var. mentagrophytes
T. m.var. mentagrophytes
T. papillosum
T. quinckeanum
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
424.63
344.79
511.73
677.86
428.63
558.66
764.84
318.56
110.65
160.66
347.55
572.75
Urease Urease
Urease
Man, arm
Man, skin, arm
Erinaceus europaeus (hedgehog)
Man, nail
Human, skin of foot
Human, skin of foot
Skin of camel
Human, deep trichophytosis
Man, pubic hair
Man, skin, arm
Human
Man, skin of leg
Haarlem, Netherlands
Netherlands
New Zealand
Germany
Netherlands
Netherlands
Saudi Arabia
Netherlands
Netherlands
Netherlands
France
Germany
Arthroderma strains
A. benhamiae (MT-)
A. benhamiae (MT)
A. simii (MT)
A. simii (MT)
CBS
CBS
CBS
CBS
807.72
808.72
417.65
448.65
Man
Man
Chicken
Poultry
Spain
South Africa
India
India
MA, macroconidia; , positive; , negative.
with several fragments of the test fungi from SGA
cultures. The test materials were incubated for 4 weeks
at 288C and hairs examined in lactophenol cotton blue
(LCB, Fluka, France) mounts at regular intervals with
a light microscope [811,16].
Pigment production on Cornmeal dextrose agar (CMDA)
and Bromcresol purple-milk solids-glucose agar (BCP-MS-G)
CMDA (Difco, Detroit, USA) medium can be used to
differentiate members of the T. mentagrophytes complex from T. rubrum on the basis of pigment production. Most T. rubrum isolates form deep wine-red
reverse pigmentation when grown on this medium,
while T. mentagrophytes complex colonies show vari– 2008 ISHAM, Medical Mycology, 46, 811822
able pigmentation ranging from uncolored to reddishbrown [8,15]. The type of growth (profuse versus
restricted) and a change in the pH indicator (BCP)
indicating alkalinity are additional useful characteristics. On BCP-MS-G (Himedia, Mumbai, India), T.
rubrum strains show restricted growth and produce no
alkaline reaction, whereas members of the T. mentagrophytes complex typically show profuse growth and
alkaline reactions [8,10,11,17,18].
Tween opacity test
The medium was prepared with 10 g peptone, 5 g of
NaCl, 0.1 g CaCl2, and 15 g of agar per liter distilled
water. After the autoclaved medium cooled to about
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
Species
814
Ates et al.
508C, 5 ml of autoclaved Tween 80 was added to it. The
Tween opacity test was considered positive when a halo
of crystals was detected macroscopically or microscopically around a colony after a period of 4 weeks [19].
Sorbitol assimilation
The medium was prepared with 3 g beef extract, 10 g
peptone, 5 g NaCl, and 1 cm3 bromthymol blue per
liter distilled water with a final pH of 7.2, then
autoclaved at 1218C for 15 min. Then a 10% sorbitol
solution was prepared and added at 1/10 to the
mentioned solvent [20].
Salt tolerance
Morphologic examination
The presence of micro- and macroconidia was investigated by inoculating a portion of growth from stock
cultures to brain heart infusion agar (BHIA), UPA,
CMDA, Lowenstein-Jensen agar (LJA), malt extract
agar (MEA), oatmeal agar (OA), Oxoid chromogenic
Candida agar (OCCA), and potato dextrose agar
(PDA). All cultures were incubated at 288C and
conidial formation was compared on the media 5, 10
and 15 days after inoculation. Morphology was examined through light microscopic examination of LCB
stained portions of growth from each of the media. All
strains were screened twice for morphological, physiological and biochemical studies.
Statistical analysis
Statistical analysis was performed using the statistical
package SPSS v 10.0 and Epi Info version 3.2. The
categorical data between groups were analysed by using
a Chi square test and Cochran’s Q test. A P value
of B0.05 was considered statistically significant.
Results
The results of urea hydrolysis in different media at
various incubation temperatures are illustrated in
Table 2. Data from the hair perforation, pigment
production, Tween opacity, and sorbitol assimilation
studies are found in Table 3. The production of microand macroconidia at each of the time points on each of
the media can be seen in Table 4a,b. The results of the
salt tolerance investigations and production of macro-
Discussion
Identification of dermatophytes is often based on
phenotypic characteristics of colonies grown in pure
culture on SGA and their microscopic morphology.
However, these criteria alone may be insufficient since
colonial features may vary within a taxon or be similar
to other fungi. Characteristic pigmentation may fail to
develop and isolates, especially Trichophyton spp., may
not sporulate. Special media may be required to induce
pigment production, as well as to obtain sporulation,
and physiologic tests in conjunction with morphological pattern could be needed for correct identification
[11].
Recently, Guoling et al. [22] reported that T. rubrum
shows a high degree of morphological diversity, including the presence or absence of reflexively branching
hyphae, micro- and macroconidia, red colony pigmentation and urease activity. It has already been shown by
microsatellite analysis that there is no group of
genotypes which correlate to a special morphotype in
T. rubrum [23]. Gräser et al. [23] noted an association
between morphological features and genotypes in two
populations, T. rubrum and T. violaceum. Phenotypically, the T. rubrum population was dominated by
strains that did not produce urease and lacked reflexively branching hyphae (97%), whereas all isolates with
reflexive hyphae were in T. violaceum. However, urease
was expressed in strains of both populations, with or
without the presence of reflexive hyphae.
Krempl-Lambrecht [24] investigated hydrolysis of
urea on urea agar and reported that 75% of T.
mentagrophytes strains were urease positive in 10
days, 5% in 20 days and 20% were negative. In contrast,
10% of T. rubrum strains were positive in 10 days, 70%
in 20 days and 20% were negative. However, Kane and
Fischer [14] noted that urea broth medium was
preferred over agar based media because it was more
sensitive. In our study, urea hydrolysis was best detected
on UPA at 288C and it was found to be statistically
superior when compared to the other methods (P B
0.01, Fig. 1, Table 2). On the other hand, it is important
to note that the usefulness of the urease test has a
certain time limit. Therefore, when using the urease
test, it should be read at 4872 hours after inoculation.
Most of the T. mentagrophytes isolates will hydrolyse
urea within this period, but T. rubrum will hydrolyse
urea more slowly and positive results will be found if
the medium is incubated for ]710 days (Table 2).
– 2008 ISHAM, Medical Mycology, 46, 811822
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
Sabouraud glucose agar was supplemented with 3%,
5%, 7% and 9% NaCl, and was used to detect speciesspecific salt tolerance, as well as the stimulation of
macroconidia formation [8,21].
conidia on SGA supplemented with 3%, 5%, 7% and
9% NaCl are in Table 5.
Species
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
100081
592.68
289.86
422.67
517.63
518.63
389.58
303.38
202.88
287.86
100084
102856
376.49
302.60
363.53
363.62
392.58
100237
424.63
344.79
511.75
677.86
428.63
558.66
764.84
318.56
110.65
160.66
347.55
572.75
807.72
808.72
417.65
448.65
228C
288C
378C
UB
USA
UPA
UB
USA
UPA
UB
USA
UPA
29, 7
19, 2
19, 2
1
3
29, 4
7 9 , 11 39, 7
29, 4
20 9
20 9
20 9
2
1
29, 5
1
39, 4
29, 9
1
29, 7
19, 3
7 9 , 11 29, 3
39, 4
29, 4
2
29, 5
2
6 9 , 10 10 9 , 24 29, 5
29, 6
29, 5
39, 5
39, 6
3 9 , 16 59, 6
49, 6
59, 6
29, 5
29, 5
19, 4
2h9 , 3 79
6 9 , 15 1
79
19, 3
59, 9
19, 6
29, 5
5 9 , 17 89,
29, 4
19, 5
5 9 , 10 17 9
7 9 , 17 79
7 9 , 17 59, 6
5 9 , 17 2 9 , 12 7 9 , 12 7 9 , 17 3 9 , 17 6 9 , 10 6 9 , 10 6 9 , 10 29, 5
29, 5
19, 3
2h9 , 3 89,
7 9 , 17 1
79
19, 3
59, 8
19, 5
29, 5
18 9
4
10 9 , 12 14 9
49, 6
4
49, 5
10 2
18 9
10 3
18 9
18 9
49, 7
13 9 ,17 16 9
16 9
10 9
2
1
1
3
3
1
3
10 9 , 12 8
29, 3
1
29, 5
19, 3
39, 5
39, 5
5 9 , 17 29, 5
6 9 , 10 5 9 , 20 9 9 , 24 19, 2
29, 5
29, 5
29, 5
5 9 , 20 19, 3
29, 5
7
39, 5
29, 5
39, 5
39, 5
39, 5
29, 3
29, 5
19, 2
2h9 , 1 19, 2
19, 2
19, 3
19, 3
29, 5
29, 5
29, 5
19, 2
7
49, 5
7
19, 4
19, 2
1
10 7
49, 8
19, 4
49, 7
39, 7
39, 5
7
4
19, 3
7
49, 7
29, 3
59, 6
59, 6
59, 6
19, 3
1
19, 2
2h9 , 1 19, 2
39, 5
1
29, 4
19, 3
49, 7
29, 4
29, 7
49
1
1
1
4
29, 7
7
2
1
16 9
18 9
18 9
1
1
29, 4
1
29, 3
29, 7
1
29, 3
2
11 29, 3
29, 3
29, 7
29, 4
49
1
1
1
7 9 , 10 49,
19, 4
29, 4
39
29, 3
39, 7
39
2
1
29, 7
19, 4
1
69, 7
69, 7
59, 6
1
1
19, 3
2h9 , 1 29, 4
1 9 ,3 1
19
19, 2
29, 3
19, 2
19, 2
79
39, 7
79
19, 2
1
1
10 19, 2
39
39
79
3
39
39
29
29, 3
69, 7
59, 6
59, 6
1
1
19, 3
2h9 , 1 29, 3
39, 4
1
1
29, 3
19, 3
29, 5
UB, urea broth; USA, urea slant agar; UPA, urea Petri agar; h, hour; 9, weak; , positive.
Conventional diagnosis of Trichophyton spp.
T. fischeri
T. fluviomunionse
T. kanei
T. kuryangei
T. kuryangei
T. kuryangei
T. megninii
T. pervesii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. rodhainii
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum var. nigricans
T. asteroides
T. erinacei
T. erinacei
T. erinacei
T. interdigitale
T. interdigitale
T. langeronii
T. mentagrophytes
T. m.var. mentagrophytes
T. m.var. mentagrophytes
T. papillosum
T. quinckeanum
A. benhamiae (MT)
A. benhamiae (MT)
A. simii (MT)
A. simii (MT)
CBS No
815
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– 2008 ISHAM, Medical Mycology, 46, 811822
Table 2 Results of urea hydrolysis in different media and incubation temperatures according to days.
816
Table 3
Ates et al.
Results of hair perforation, pigment production, Tween opacity and sorbitol assimilation tests according to days.
CBS No
BCH
T. fischeri
T. fluviomunionse
T. kanei
T. kuryangei
T. kuryangei
T. kuryangei
T. megninii
T. pervesii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. rodhainii
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum var. nigricans
T. asteroides
T. erinacei
T. erinacei
T. erinacei
T. interdigitale
T. interdigitale
T. langeronii
T. mentagrophytes
T. m.var. mentagrophytes
T. m.var. mentagrophytes
T. papillosum
T. quinckeanum
A. benhamiae (MT)
A. benhamiae (MT)
A. simii (MT)
A. simii (MT)
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
100081
592.68
289.86
422.67
517.63
518.63
389.58
303.38
202.88
287.86
100084
102856
376.49
302.60
363.53
363.62
392.58
100237
424.63
344.79
511.73
677.86
428.63
558.66
764.84
318.56
110.65
160.66
347.55
572.75
807.72
808.72
417.65
448.65
5
52, 6 6
53, 6 6
7
221
5
5
6
10 6
5
53,6 6
SH
131
51, 6 6
64,7 55, 6 152, 21 8
5
11 14 131
CMDA
BCP-MS-Gr
BCP-MS-Gy
12
10
10
7
7
7
12
10
12
10
7
10
10
29,
2
29,
39,
59,
29,
29,
29,
29,
29,
29,
29,
29,
29,
29,
29,
3
2
3
3
3
3
5
2
4
4
3
2
3
3
3
5
4
5
7
3
3
3
3
5
3
3
3
3
3
3
BCP-MS-Gp TO
SA
8
5 9,
5 9,
7
8
5 9,
5
59,
5 9,
5 9,
5 9,
5 9,
5 9,
5 9,
5 9,
5 9,
5 9,
5 9,
4
4
4
4
4
29,
29,
4
4
4
4
4
5
5
5
5
7
79,
7
7
7
7
7
7
7
7
79,
39,
39,
39,
7
79,
7
7
7
7
7
7
7
7
79,
8
8
8
8
8
8
8
8
8
8
8
8
8
8
4
5
3
5
5
8
8
4
3
4
4
3
3
6
3
3
4
3
3
3
3
3
3
5
3
3
3
3
3
2
3
2
10 10 4
4
4
10 10 BCH, blond child hair; SH, sheep hair; n19: number of perforation was between 1 to 9; n]10: number of perforation was ]10.
CMDA, Cornmeal dextrose agar; BCP-MS-G, Bromcresol purple-milk solids-glucose agar; r, red; y, yellow; p, purple; 9, weak; , positive.
TO, Tween opacity test; SA, sorbitol assimilation.
Takahashi et al. [25] studied urease activity of 7
Japanese, 5 Kenyans and 6 European and New Zealand
(including the CBS strains used in this study) T.
erinacei isolates on Christensen’s urea agar slant at
258C for 7 days. The authors noted that only one of the
Japanese isolates, and all of the Kenyans were ureasepositive, whereas the Europeans strains (except CBS
108.91) were urease-negative. However, we observed
that two of the Europeans (CBS 344.79 and CBS
677.86) and one New Zealand isolates (CBS 511.73)
were urease positive on the same medium after 36 days
of incubation at 228C and 288C. These results suggest
that the urease activity of this dermatophyte is highly
variable.
Sinski et al. [26] observed that the most reliable test
for the differentiation of T. mentagrophytes from T.
rubrum was the standard in vitro hair perforation test.
While it is effective and easy-to-perform, hair perforation is time-consuming, requiring 1 to 4 weeks to
complete. Although in our study more perforation was
found with hair from blond children, the results of the
tests using hair from this source and sheep were not
statistically significant (P 0.05, Fig. 2, Table 3). While
the test was also performed with goat hair, the dark
color of this hair under light microscope interfered with
the examination.
Summerbell et al. [18] reported that T. mentagrophytes isolates had; (1) diffuse growth with a strong
alkaline reaction visible within 7 days on BCP-MS-G,
(2) a strong positive urease capability, and (3)
perforated hair. On the other hand, T. rubrum strains
were urease negative, did not perforate hair, and
produced no alkalinity on BCP-MS-G after 7 days.
Furthermore, the authors noted that T. rubrum with
– 2008 ISHAM, Medical Mycology, 46, 811822
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
Species
Conventional diagnosis of Trichophyton spp.
817
Table 4a Production of micro- and macroconidia according to culture media on days 5, 10, and 15.
Species
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
100081
592.68
289.86
422.67
517.63
518.63
389.58
303.38
202.88
287.86
100084
102856
376.49
302.60
363.53
363.62
392.58
100237
424.63
344.79
511.73
677.86
428.63
558.66
764.84
318.56
110.65
160.66
347.55
572.75
807.72
808.72
417.65
448.65
PDA
BHIA
MEA
LJA
MI
MA
MI
MA
MI
MA
MI
MA
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
/ /10
//
/ /
/ / /
//
/8/10
30//
//
//
//
//
/ /
//
//
/ /
//
/ /
//
//
//
//
/ /
//
//
/ /
//
//
/ /
20//
//
//
//
6/10/10
/ /
10//
/ /
/ /5
/ /
/ /
/ /
/ /3
6/8/10
/10/10
//
20//
/ /
/ /
/ /
/ /
/2/2
/ /
/ /
1/1/1
2*/1*/2*
1/2/1
/ /
3/4/4
/ /
/ /
/ /
/ /2
/ /
/ /
/ /
/ /2*
/20/
/ /
20/20/20
//
/ /
//
/ /
//
//
//
//
//
//
//
10/25/
//
//
//
//
/30/
//
//
//
15//
//
//
//
//
//
//
//
//
//
//
//
//
//
//
10/20/
//3
//
//
2/5/5
1/2/5
8/10/10
5/15/15
//
//
2/2/2
4/7/
//4
//
10/15/
/1/5
//
2/2/5
//
1/3/10
/2/3
2/2/3
//
2/2/6
//
8/8/10
//
1/4/5
//
/1*/4*
4/4/6
//
//
//
// //
/10/24
10/15/15
8/10/10
/20/30
//
//
//
//
/20/25
//
//
// //
10/20/24
//
//
20//
20//
//
//
//
//
//
//
//
//
//
//
//
//
// //
8/10/10
// /2*/
// /1/1
// //
1/2/4
//
//
// 3/4/
//2
//
// /1/2
// /2/1
/1*/5*
/2*/3*
/1/2
2/2/3
1/2/2
// // 4/8/8
// // // /1*/3*
1/2/2
/1/2
//
//
/ / //
/ /
//
//
//
//
//
//
//
20//
//
//
//
//
20//
//
//
//
//
//
//
//
//
//
//
//
//
//
//
//
//
/ /
//
15//
/2/3
/ /1
/ /
5/6/6
3/3/5
8/10/10
//
//
//
3/3/3
6/7/
1/1/4
//
10/15/
1/2/5
1/3/3
2/8/10
/6/6
//
/2/3
3/3/3
//
4/4/7
/ /2*
10/10/12
/ /
4/5/5
/ /
/1*/4*
4/4/6
/ /
PDA, Potato dextrose agar; BHIA, Brain heart infusion agar; MEA, Malt extract agar; LJA, Lowenstein-Jensen agar; MI, microconidia; MA,
macroconidia; , 30 ; *, In all microscopic field; , negative.
kanei, fischeri and raubitschekii morphotypes all
showed a pattern of restricted growth, rapid development of red reverse pigmentation, and were alkaline positive on BCP-MS-G.
In this present study, while most of the T. rubrum
strains produced (72.2%) red pigment on CMDA,
members of the T. mentagrophytes complex and Arthroderma strains did not. Although this difference was
found to be statistically significant (P B0.01, Table 3),
it should be again noted that only the majority of T.
rubrum strains produced pigment. Our suggestion is
that pigment production is not a reliable morphological
character. In this investigation, somewhat better pigment formation was noted with BCP-MS-G medium.
– 2008 ISHAM, Medical Mycology, 46, 811822
Most of the T. rubrum strains (88.8%) produced red
pigment on BCP-MS-G, except megninii (uncolored,
5.6%) and pervesii (yellow, 5.6%) morphotypes. The
current study has also demonstrated that features
thought to be characteristic for a morphotype (e.g.,
fischeri or megninii) were not expressed (Table 3).
Our present observations are in agreement with those
of Kane and Fischer [27] who reported that T. rubrum
with megninii morphotype produced a spreading velvety growth. A darkening of the BCP milk dextrose agar
indicated an increase in the pH and no reddish color
was prominent around the colony. These authors noted
that the aerial growth may show some pink color as the
colony ages [27], while in our study, we found that most
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
T. fischeri
T. fluviomunionse
T. kanei
T. kuryangei
T. kuryangei
T. kuryangei
T. megninii
T. pervesii
T. raubitschekii
T .raubitschekii
T. raubitschekii
T. raubitschekii
T. rodhainii
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum var. nigricans
T. asteroides
T. erinacei
T. erinacei
T. erinacei
T. interdigitale
T. interdigitale
T. langeronii
T. mentagrophytes
T. m.var. mentagrophytes
T. m.var. mentagrophytes
T. papillosum
T. quinckeanum
A. benhamiae (MT)
A. benhamiae (MT)
A. simii (MT)
A. simii (MT)
CBS No
818
Ates et al.
Table 4b Production of micro- and macroconidia according to culture media and on days 5, 10, and 15.
Species
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
100081
592.68
289.86
422.67
517.63
518.63
389.58
303.38
202.88
287.86
100084
102856
376.49
302.60
363.53
363.62
392.58
100237
424.63
344.79
511.73
677.86
428.63
558.66
764.84
318.56
110.65
160.66
347.55
572.75
807.72
808.72
417.65
448.65
OA
OCCA
CMDA
UPA
MI
MA
MI
MA
MI
MA
MI
MA
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
5/10/15
/10/6
//
/ /
//
//
/ /
//
//
/ /
//
//
//
20/30/30
//
/ /
//
//
//
//
30//
//
//
10/15/
20/30/30
10//
20//
//
10//
/ /
6//
10//
//
//
//
// //
3*// /1*/
//7
// //1
/2/2
/5*/
4/10/ /10/15
3/ / 2/4/8
// // // 4/4/
// 5*// /1/1
// 1/2/1
/2*/
// // // 5*// // // // // // 6//
// //
10//
//
//
//
//
//
//
//10
//
//
//
/10/10
//
//
//
//
//20
//
//
//
//
/10/
//
10/10/
10/8/10
//
20//
//
//
//
//
//
//
/ /
10//
/ /7
/ /
/ /
/ /2*
/ /
/ /3
/ /
//
/15/20
7/20/
1/2/2
/ /
/ /
/1/2
/ /
/ /
/ /1*
4//
/ /2*
/ /
/ /
/2*/2*
1/3/3
/ /
5/10/10
/ /
/ /2*
/1*/
/ /3*
/ /
1*/8*/8*
/5*/5*
//10
//
// // /5/6
// //
8/10/
// //
//
//
/8/
24/20/20
// //
//
8/10/20
//
//
20//
//
20/20/20
8/ / // 5/5/
//
//
// 30//
// // //
8/10/
// 6/8/8
// // // // // // // /3/4
5/8/8
2*// // // // // // // // //2
// // // // // //2
2*// // // // // // 1/2/2
// / /10
6/8/8
/ /
/ /
//
/ /
/20/
5/ /
/10/10
//
/20/
/ /
/ /
//
/8/
20//
10//
/ /
//
20//
//
20/20/20
8/10/10
//
8/8/4
/ /
16//
//
/ /
10/10/
/ /
10/10/
//
//
/ / / /3*
5/12/
/ / / / / / / / / / / / /2/2
/10/
/3/4
/ / /3*/ / / 5/10/10
/ /1*
/ / / /2*
/ / / / / / / / /2*/ 2*// / / /4/4
/ / / / /1*/ / / / / 2*/1*/2*
/ / OA, Oat meal agar; OCCA, Oxoid chromogenic Candida agar; CMDA, Cornmeal dextrose agar; UPA, Urease Petri agar; MI, Microconidia;
MA, Macroconidia; , 30 ; *, In all microscopic field; , positive; , negative.
of the T. mentagrophytes complex (83.3%) and Arthroderma strains (100%) produced yellowish pigment. We
observed that the growth of T. mentagrophytes complex
and Arthroderma strains on BCP-MS-G caused an
increase in the pH within a week of inoculation.
However, such a pH change was observed around the
colonies of T. rubrum after 5 days, and on all media
after 8 days (Table 3).
Slifkin and Cumbie [19] noted in Tween opacity
studies that all the isolates of T. rubrum tested produced
macroscopic halos around the colonies 57 days after
inoculation, while T. mentagrophytes var. interdigitale
strains formed visible halos after 1620 days of incubation. However, in our study, the efficacy of the Tween
opacity test was not found to be statistically significant
in differentiating between the two taxa (P 0.05,
Table 3). Rezusta et al. [28] reported that 147 T.
mentagrophytes strains were able to assimilate sorbitol
in API 20C AUX strips when read after 7 days of
incubation at 30 8C, whereas 36 T. rubrum strains did
not. However, in our study, the sorbitol assimilation
test failed to distinguish both taxa from each other
(P 0.05, Table 3).
Yucel and Al-Ali [29] found that 69 T. mentagrophytes strains formed macroconidia on LJA (98.5%),
Pai (96%), Lactrimel (63%), and SGA (25%) media. In
line with their study, we demonstrated that the production of macroconidia was best on LJA after 15 days of
– 2008 ISHAM, Medical Mycology, 46, 811822
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T. fischeri
T. fluviomunionse
T. kanei
T. kuryangei
T. kuryangei
T. kuryangei
T. megninii
T. pervesii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. rodhainii
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum var. nigricans
T. asteroides
T. erinacei
T. erinacei
T. erinacei
T. interdigitale
T. interdigitale
T. langeronii
T. mentagrophytes
T. m.var. mentagrophytes
T. m.var. mentagrophytes
T. papillosum
T. quinckeanum
A. benhamiae (MT)
A. benhamiae (MT)
A. simii (MT)
A. simii (MT)
CBS No
Conventional diagnosis of Trichophyton spp.
Table 5
819
Species-specific growth in SGA supplemented with 3%, 5%, 7% and 9% NaCl.
CBS No
3%
5%
7%
9%
T. fischeri
T. fluviomunionse
T. kanei
T. kuryangei
T. kuryangei
T. kuryangei
T. megninii
T. pervesii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. raubitschekii
T. rodhainii
T. rubrum
T. rubrum
T. rubrum
T. rubrum
T. rubrum var. nigricans
T. asteroides
T. erinacei
T. erinacei
T. erinacei
T. interdigitale
T. interdigitale
T. langeronii
T. mentagrophytes
T. m.var. mentagrophytes
T. m.var. mentagrophytes
T. papillosum
T. quinckeanum
A. benhamiae (MT)
A. benhamiae (MT)
A. simii (MT)
A. simii (MT)
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
CBS
100081
592.68
289.86
422.67
517.63
518.63
389.58
303.38
202.88
287.86
100084
102856
376.49
302.60
363.53
363.62
392.58
100237
424.63
344.79
511.73
677.86
428.63
558.66
764.84
318.56
110.65
160.66
347.55
572.75
807.72
808.72
417.65
448.65
, positive; , negative.
inoculation (P B0.001, Fig. 3, Table 4a). Our results
also revealed that both LJA and BHIA supported rich
sporulation of micro- and macroconidia, and these two
media might be used as routine mycological media like
UPA (Figs. 4 and 5).
Kane et al. [8] reported that T. rubrum strains with
fischeri, kanei, megninii, and raubitschekii morphotypes
had strong, restricted growth on 35% NaCl-supplemented SGA and produced no conidia. In our study,
the growth in NaCl concentrations was found to be
statistically significant for 3% NaCl (P 0.001). It was
observed that the salt tolerance test was not as effective
in differentiating the two taxa, as well as the two related
species and morphotypes (P 0.05, Table 5).
In the present study, we critically evaluated the value
of conventional diagnostic tests in the identification of
dermatophyte species as summarized in Tables 24a,b,
and 5. Several of the isolates included in our investigation, i.e., T. fischeri CBS 100081, T. fluviomunionse CBS
– 2008 ISHAM, Medical Mycology, 46, 811822
592.68, T. kanei CBS 289.86, T. kuryangei CBS 422.67,
T. kuryangei CBS 517.63, T. raubitschekii CBS 287.86,
T. raubitschekii CBS 100084, T. rodhainii CBS 376.49,
T. rubrum CBS 392.58, T. pervesii CBS 303.38, T.
rubrum var. nigricans CBS 100237 (ATU TR9) were
evaluated by Gräser et al. [4]. The results in the latter
for urea hydrolysis in urea broth, hair perforation, and
the production of micro- and macroconidia on MEA
are similar to those we found (Tables 2 and 3, and 4a).
Dr Julius Kane [30] proposed T. fischeri in 1977
based on studies of primary clinical isolates which
readily formed micro- and macroconidia. Gräser et al.
[4] investigated 2 isolates of the organism (CBS 288.86
and CBS 100081) after 23 years of continuous culturing
and could not find macroconidial production in either
strain. Our results with CBS 100081 correspond to
those of Gräser et al. [4] (Table 4a,b). It is a well known
fact that majority of dermatophyte species maintained
under laboratory conditions for a longer period of time
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
Species
820
Ates et al.
Fig. 3 Trichophyton mentagrophytes var. mentagrophytes CBS 110.65
at 288C in Lowenstein-Jensen agar after 15 days.
loose their ability to form conidia, especially macroconidia. The mating types of Arthroderma spp. maintained under ideal laboratory conditions have been
found to lose their mating abilities.
In another study by Gräser et al. [3], isolates of
T. interdigitale CBS 558.66, T. interdigitale CBS 428.63,
T. mentagrophytes CBS 318.56, T. papillosum CBS
347.55, A. simii (MT-) CBS 417.65, A. simii (MT)
CBS 448.65 were investigated for urea hydrolysis in
Christensen’s urea broth and hair perforation test.
Their results are very similar to those noted in this
study (Tables 2 and 3). To the best of our knowledge,
our investigations are the first to discuss the morphological, physiological and biochemical characteristics of
Trichophyton kuryangei CBS 518.63, T. megninii CBS
389.58, T. raubitschekii, CBS 202.88, T. raubitschekii
CBS 102856, T. rubrum CBS 302.60, T. rubrum CBS
363.53, T. rubrum CBS 363.62, T. asteroides CBS
424.63, T. langeronii CBS 764.84, T. mentagrophytes
var. mentagrophytes CBS 110.65, T. mentagrophytes var.
mentagrophytes CBS 160.66, T. quinckeanum CBS
572.75, A. benhamiae (MT ) CBS 807.72, and A.
benhamiae (MT) CBS 808.72.
Summerbell et al. [18] obtained five A. benhamiae
strains from Centers for Disease Control, Atlanta,
Georgia and two A. vanbreuseghemii isolates from
Department of Health, New York, which produced a
strongly positive urea hydrolysis, perforated hair, and
showed rapid and diffuse growth with a concomitant
moderate-to-strong alkaline reaction visible within 7
days on BCP-MS-G. We obtained parallel results with
these fungi as well as A. simii (Tables 2 and 3).
While some dermatophyte strains have been sequenced to allow for their accurate identification,
most isolates of T. mentagrophytes sensu lato, i.e., in
the CBS collection database, need to be sequenced to
ensure that they have been appropriately identified.
Hence, the classification is currently in a state of change
and the identities of many isolates are not yet settled
(R. C. Summerbell, pers. comm.). This suggests that
more data from additional studies are required to
enhance the validity of findings obtained from this
study.
Finally, conventional procedures are still important
for laboratory diagnosis. This study underlines the
practical, simple and easy-to-perform tests, for species
identification of dermatophyte strains that are conducted by a large number of laboratories. The results of
this study suggests that; (i) optimum urea hydrolysis
test results were detected on UPA incubated at 288C
(P B0.01), (ii) better sporulation was provided by LJA
and BHIA incubated for 15 days (P B0.001), (iii)
highest pigment formation was noted on CMDA
(P B0.01) and BCP-MS-G media, might yield reliable
differentiation. We conclude that the media mentioned
Fig. 2 Hair perforation (blond child) by
Trichophyton mentagrophytes var. mentagrophytes CBS 110.65 at 288C after 5 days.
– 2008 ISHAM, Medical Mycology, 46, 811822
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
Fig. 1 Culture of Trichophyton raubitschekii CBS 102.856 at 288C in
urease Petri agar after 3 days.
Conventional diagnosis of Trichophyton spp.
821
sharing with us her most valuable works. Our special
thanks go to the anonymous reviewers whose valuable
comments helped us to improve the readability and
scientific content of this manuscript.
Declaration of interest: The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of the paper.
References
Fig. 4 Typical Trichophyton-macroconidium (arrow) formed by
Trichophyton rubrum CBS 363.62 at 288C in brain heart infusion
agar after 15 days (Lactophenol cotton blue 400).
above are highly efficacious in facilitating the identification of these two taxa based on the basis of their
morphological and physiological characteristics.
Acknowledgements
This study was supported by the Resarch Fund of
Cukurova University (Project No: TF2004D7). We are
indebted to Dr Richard C. Summerbell from Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, for his conducive comments on the CBS strains,
and valuable suggestions on the current taxonomy of
dermatophyte fungi. We also thank PD Dr Yvonne
Gräser from the Institute of Microbiology and Hygiene
(Charité), Humboldt University, Berlin, Germany, for
– 2008 ISHAM, Medical Mycology, 46, 811822
Downloaded from http://mmy.oxfordjournals.org/ by guest on January 3, 2017
Fig. 5 Macroconidium (arrow) formed by Arthroderma simii CBS
417.65 (MT-) at 288C in Lowenstein-Jensen agar after 15 days
(Lactophenol cotton blue 400).
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